Rotavirus single stranded (ss) RNA probes prepared by in vitro transcription of purified virions were used in a dot hybridizaton assay to detect the presence of rotavirus in stools and other biological materials. The assay is highly specific and sensitive and its use may facilitate epidemiological studies of rotavirus gastroenteritis. RNA transcription probes have also been employed successfully in analyzing and quantitating the degree of genetic relatedness among different rotavirus strains of human and animal origin. Probes prepared from rotavirus cDNA cloned into pBR322 have also been used in the dot hybridization assay. They have allowed the recognition of specific rotavirus sequences corresponding to discrete genes. Application of these gene-specific probes will be useful in epidemiological studies aimed at the identification of rotavirus serotypes and other markers.